Clonal architecture of CXCR4 WHIM-like mutations in Waldenström Macroglobulinaemia

CXCR4^WHIM somatic mutations are distinctive to Waldenström Macroglobulinaemia (WM), and impact disease presentation and treatment outcome. The clonal architecture of CXCR4^WHIM mutations remains to be delineated. We developed highly sensitive allele-specific polymerase chain reaction (AS-PCR) assays for detecting the most common CXCR4^WHIM mutations (CXCR4^{S338X C>A and C>G}) in WM. The AS-PCR assays detected CXCR4^S338X mutations in WM and IgM monoclonal gammopathy of unknown significance (MGUS) patients not revealed by Sanger sequencing. By combined AS-PCR and Sanger sequencing, CXCR4^WHIM mutations were identified in 44/102 (43%), 21/62 (34%), 2/12 (17%) and 1/20 (5%) untreated WM, previously treated WM, IgM MGUS and marginal zone lymphoma patients, respectively, but no chronic lymphocytic leukaemia, multiple myeloma, non-IgM MGUS patients or healthy donors. Cancer cell fraction analysis in WM and IgM MGUS patients showed CXCR4^S338X mutations were primarily subclonal, with highly variable clonal distribution (median 35·1%, range 1·2-97·5%). Combined AS-PCR and Sanger sequencing revealed multiple CXCR4^WHIM mutations in many individual WM patients, including homozygous and compound heterozygous mutations validated by deep RNA sequencing. The findings show that CXCR4^WHIM mutations are more common in WM than previously revealed, and are primarily subclonal, supporting their acquisition after MYD88^L265P in WM oncogenesis. The presence of multiple CXCR4^WHIM mutations within individual WM patients may be indicative of targeted CXCR4 genomic instability.